PT1224314E - Lentiviral triplex dna, and vectors and recombinant cells containing lentiviral triplex dna - Google Patents

Abstract

The present invention provides nucleic acid, vectors, viruses, and recombinant cells comprising triple-stranded structures, such as those resulting from central initiation and termination of HIV-1 reverse transcription at the center of HIV-1 linear DNA genomes. These triplex structures can act as a cis-determinant of HIV-1 DNA nuclear import, allowing infection of non-dividing target cells. In one aspect, the presence of the DNA triplex sequence in an HIV vector strongly stimulates gene transfer in hematopoietic stem cells. The invention also provides methods of using these triplex structures for making recombinant cells, as well as methods of using the recombinant cells to express proteins of interest both in vitro and in vivo.

Description

DESCRIPTION

LENTIVIRAL TRIPLEX DNA AND VECTORS AND RECOMBINANT CELLS CONTAINING LENTIVIRAL TRIPLEX DNA

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to the field of biotechnology, and especially to viral nucleic acids and cells containing viral nucleic acids. More particularly, the present invention relates to viral nucleic acid sequences which may form part of triplex DNA structures as well as to nucleic acid vectors, viruses, and cells containing these viral nucleic acid sequences. It further relates to methods of using such DNA structures and nucleic acid sequences.

Description of Related Art Gene transfer in hematopoietic stem cells (HSCs) has great potential both for gene therapy of hereditary diseases as well as acquired diseases and for understanding mechanisms that regulate normal and pathological hematopoiesis. Since HSCs have extensive proliferative abilities, stable gene transfer should include the genomic integration of the transgene. Moloney murine leukemia virus (MoMLV) -based retroviruses have been very popular because they integrate into host cell genomes and may allow long-term transgene expression. However, these oncoretroviruses do not integrate into cells without division, and most HSCs are quiescent. Many prestimulation protocols using different cytokine associations have been developed to activate the cycles of the HSCs in order to make them transducable by oncovirus-derived and mitotic-dependent gene transfer vectors. However, stimulation of cytokine 1 induces differentiation in conjunction with proliferation and the potentialities of HSC can be lost during the transduction process. This problem can be overcome by using lentivirus-derived vectors since lentiviruses have been shown to infect both dividing and non-dividing cells (Poznansky et al., 1991; Naldini et al., 1996). Initial disclosures published in the last three years using such lentivirus-derived vectors for HSC transduction have shown promising but very heterogeneous results (Case et al., 1999; Evans et al., 1999; Uchida et al., 1998; Miyoshi et al. , 1999).

Lentiviruses have developed a nuclear import strategy that allows their genomic DNA to traverse the nuclear membrane of a host cell. This active nuclear import of lentivirus is responsible for its unique ability, among retroviruses, to replicate effectively in non-dividing target cells, such as macrophages tissue. The restriction of replication of oncoviruses such as MoMLV to divide cells seems to be due to the requirement to disrupt the nuclear membrane barrier during mitosis, allowing MoMLV preintegration complexes (PICs) to enter the nucleus (Roe et al., 1993) . Independent replication of lentivirus mitosis, in the origin of its replication strategies in vivo and consequently of its pathogenicity, also allowed the generation of lentiviral gene transfer vectors with promising therapeutic applications (Poznansky et al., 1991; Naldini et al. , 1996). Independent replication of lentivirus mitosis was initially demonstrated by the productive infection of nonmitotic chondrocyte cells by VISNA lentivirus (Thormar, 1963). Soon after its discovery, HIV was shown to replicate in differentiated primary macrophages (Gartner et al., 1986; Ho et al., 1986). HIV DNA integrates into the chromatin of non-mitotic target cells (Weinberg et al., 1991; Lewis et al., 1992), implying that HIV-1 PICs are able to cross the nuclear membrane of host cells (Bukrinsky et al. al., 1992). Thus, nuclear import independent of mitosis is an essential event responsible for the ability of lentivirus to replicate in non-dividing cells. The search for the viral determinants responsible for the active nuclear import of DNA from the HIV-1 genome was an active but controversial field of investigation. The presence of putative nuclear localization signals (NLSs) within the matrix (MA) and Vpr viral proteins led to the proposition that they could act in a redundant way on nuclear import of HIV-1 DNA (Bukrinsky et al., 1993b; Emerman et al., 1994, Popov et al., 1998, von Schwedler et al., 1994). It has been proposed that phosphorylation of a small subset (1%) of MA molecules in an exterminine tyrosine residue activates its plasma membrane release and its association with HIV-1 integrase protein (Gallay et al., 1995a, 1995b) . The contribution of these proteins to the cariophilic properties of HIV-1 PICs is currently a matter of heated debate (Freed and Martin, 1994; Freed et al., 1995; Fouchier et al., 1997; Freed et al., 1997; and Schuitemaker, 1999). More recently, NLS motifs in the integrase protein (IN) have been identified and mutations in these motifs have been reported to abolish the interaction of IN with karyopherin a, a cellular NLS receptor (Gallay et al., 1997).

SUMMARY OF THE INVENTION

Whatever the role of these candidate viral proteins in nuclear HIV importation, the present invention shows that the retrotranscribed HIV-1 genome alone produces a cis-action determinant for its nuclear import. The invention provides a nucleic acid comprising a triple-stranded structure (triplex), such as one of a lentivirus. The triplex stimulates the entry of nucleic acids into the cell nucleus. The nucleic acid may contain the cis-cPPT and CTS-action sequences of a lentivirus. The lentivirus may be any lentivirus, including, but not limited to, HIV-1, HIV-2, VISNA, EINV, FIV, and CAE. In embodiments, the nucleic acid is in the context of a vector, such as an expression vector.

Thus, the invention also provides a vector, for example, a nucleic acid vector. The nucleic acid vector may include sequences of any vector known to the skilled person in the art of utility for the transfer of nucleic acids into cells or for expression of nucleic acids in vitro. The invention further provides viruses and cells (eukaryotic and prokaryotic) containing the nucleic acid of the invention. The cells may be recombinant cells. The invention further provides a method of transferring nucleic acid to a host cell nucleus, for example, by exposing the host cell to the nucleic acid, vector, virus, or cell of the invention, to provide a recombinant cell. The method of the invention allows high-efficiency transfer of nucleic acids to the host cell nucleus, such as the nucleus of a hematopoietic stem cell. High efficiency transfer enables the skilled person to consider the expression vector of the invention for use as a therapeutic compound, particularly in gene therapy. Gene therapy can generally be used to treat blood disease, brain disease, viral disease as well as many other inherited or acquired diseases.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows that central initiation of reverse transcription is an important step in the replication cycle of HIV-1. (A) Mutations introduced into the cPPT sequence of HIV-1. Conservative and semi-conservative cPPT mutant viruses were constructed. The semi-conservative mutant cPPT-D contains 10 mutations in the 19-mer cPPT. The cPPT-AG mutant is its control virus in which a single purine mutation by purine introduces the same amino acid change coding region of the overlap integrase. Mutations are shown in inverted type. (B) Impact of cPPT mutations on HIV-1 infectivity. Viral replication kinetics in PHA-stimulated PBLs (left panel) and MT4 (right panel) cells. The cells were infected with equivalent amounts of virus particles according to the capsid (p24) antigen contents of the viral supernatants. Virus production was followed over time by RT activity. (C) Single cycle virus titrations on aphidicolin treated P4 cells with division or non-division (HeLa CD4-LTR LacZ). Β-galactosidase activity was measured using a chemiluminescence assay. Results are expressed as inoculum relative light units (RLU) / sec / ng p24, mean ± SD of four independent experiments. Figure 2 illustrates mutations in cPPT that does not affect viral production, viral DNA synthesis nor the ability of viral DNA to integrate in vitro. (A) Effect of cPPT mutations on viral production. HeLa cells were transiently transfected with plasmids pLAI, pcPPT-AG, or pcPPT-D. Viral production was measured by quantification of p24 viral antigen in cell supernatants, 48 hours post-transfection. 5 (B) Effect of cPPT mutations on reverse transcription efficiency. P4 cells were infected with the same amounts of virus particles (300 ng p24 antigen per 106 cells) and the DNA was extracted 12 hours later. Total amounts of reverse transcription viral DNA, represented by an internal HIV-1 MscI fragment, was detected by Southern blot using the same DNA fragment as a probe and quantified using a &quot; phosphorimager &quot;. (C) Effect of cPPT mutations on in vitro integration. MT4 cells were co-cultured with chronically infected H9-LAI or H9-cPPT-225 cells. In vitro integration of viral preintegration complexes, isolated from the cytoplasm of infected cells, was performed as previously described (Farnet and Haseltine, 1990). Each band is loaded with cytoplasmic DNA from 2χ10β infected cells. Figure 3 shows that the central triplex DNA mutant viruses are deficient in the nuclear import of viral DNA. (A) Strategy for the quantitative follow-up of the synthesis, circularization, and integration of HIV-1 DNA. DNA from infected cells was extracted at various times postinfection, digested with MscI and Xhol, and analyzed by Southern blot using a probe overlapping the 5'MscI site. The internal 1.9 kb DNA fragment, common to all viral DNA species regardless of their integrated or non-integrated state, indicates the total amount of viral DNA in infected cells. The 2.6 kb, 2.8 kb, and 3.4 kb signals respectively corresponding to non-integrated linear DNA, one, and two circular DNA LTRs are revealed. Since the PCR-generated probe exactly overlaps the MscI site, the intensity of each band is directly proportional to the amount of the corresponding viral DNA species. Thus, the amount of integrated proviral DNA can be calculated by subtracting from the total amount of viral DNA the signals of non-integrated and circular linear viral DNA. 6 (B) Southern blot analysis of viral DNA processed in infected cells. P4 cells were infected with equivalent amounts of each virus, normalized at the p24 levels of the supernatants. Infected cells were lysed at different post-infection times, extracted DNA, and used for the quantitative analysis described above. (C) Profiles of intracellular viral DNA, at the conclusion of one cycle of infection (48 hours postinfection). The results are expressed as percentages of total viral DNA. Similar intracellular viral DNA profiles were obtained using MT4 cells (data not shown). Figure 4 shows that the linear DNA of central triplex DNA mutant viruses accumulates in the vicinity of the nuclear membrane. (A) Core / cytoplasmic fractionation of infected P4 cells. Southern blot analysis of viral DNA from nuclear (N) and cytoplasmic (C) fractions, 24 hours post infection. Fractionation based on triton lysis was performed as previously described (Belgrader et al., 1991). DNA was restricted with MscI and hybridized using the MscI site overlay probe. (B) Detection of individual HIV-1 genomes by In situ hybridization of Fluorescence (FISH). P4 cells were infected at high multiplicity (2æg of p24 antigen per 106 cells), and hybridized using a full-length HIV-1 genome probe. Fluorescent signals were amplified by precipitation of tyramide. Optical sections through the cells were analyzed by deconvolution microscopy. Figure 5 shows that insertion of the central triplex DNA into an HIV-1 based vector enhances GFP transduction and nuclear import of vector genomic DNA. 7 (A) Schematic diagram of the genomes of the vectors. CPPT and CTS sequences of the HIV-1 genome, responsible for the formation of the triplex DNA during reverse transcription, were inserted at a central position in the HR GFP vector previously described (Naldini et al., 1996). TRIPinv-GFP includes the central triplex DNA sequence in the reverse, non-functional orientation. (B) Comparative efficiency of GFP transduction using HIV vectors with or without a central triplex DNA. HeLa cells (treated with aphidicolin) with or without division were used as targets. GFP fluorescence was quantified 48 hours post-transduction using a microplate fluorometer (Wallac). The results are expressed as the mean ± SD of a representative experiment performed in triplicate. Pseudotransduction of GFP activity was subtracted from the signal. (C) Southern blot analysis of DNA vector processed in transduced cells. Transduced HeLa cells were lysed at different post-infection times, extracted DNA, restricted, and Southern blotted using a similar strategy as for viruses. Digestion by MscI is replaced by EcoNI and Avall, and the probe is a PCR DNA fragment that exactly overlaps the EcoNI site. (D) Quantitative analysis of the intracellular state of the DNA vector, 48 hours post infection. The results are presented as percentages of total DNA vector. Figure 6 illustrates a model for nuclear import of the triplex DNA-dependent HIV-1 genome. (A) Evaluation of observed phenotypes of central triplex DNA mutant viruses. The central initiation and termination steps of HIV-1 reverse transcription create a long positive stranded DNA flap structure: the central triplex DNA. The long positive strand of HIV-1 is synthesized as two discrete half-genome segments. A downstream segment is initiated in a central copy of the polypurine tract sequence (cPPT). The upstream segment terminates downstream of the cPPT, following a long-chain displacement event of 99 nucleotides, blocked by the central termination sequence (CTS). At the conclusion of a single infection cycle viral DNA from wild-type virus is virtually completely processed into integrated provirus (* 55%), 1LTR (* 35%), and circular DNA 2LTRs (<5%), while a small fraction remains as linear DNA (<10%). Mutations in cPPT affect the formation of central triplex DNA. The final reverse transcription product of a central initiating mutant virus is a continuous double-stranded linear DNA lacking the central triplex DNA (Charneau et al., 1992). Viral DNA of the cPPT-D mutant virus accumulates in infected cells as linear DNA molecules, and is located in the vicinity of the nuclear membrane. (B) Two speculative mechanisms for nuclear import of the triplex-dependent HIV-1 genome by maturation of the reverse transcription complex (RTC) in a preintegration complex (PIC) and linear DNA translocation through the nuclear pore. Reverse transcription of HIV-1 probably occurs within a structured (ordered) complex surrounded by a set of capsid proteins. The size of the RTC exceeds the exclusion diameter of the nuclear pore. Prior to translocation, RTC undergoes maturation at a smaller ICP with loss of capsid proteins (Karageorgos et al., 1993). The formation of the triplex DNA signals the completion of viral DNA synthesis and may signal the escape of the HIV DNA from the capsid assembly. In PIC, the ends of linear DNA are likely to cross together following dimerization of integrase proteins bound to the ends of LTRs (Miller et al., 1997). The triplex DNA being in a central position, a logical structure for HIV-1 PIC would be a double strand, symmetrically folded on either side of the central triplex by the dimerization of the integrase. The triplex DNA could then constitute a vertex that could interact with 9 caryophilic transport proteins, allowing passage of the HIV-1 DNA strand through the nuclear pore. In cPPT mutant viruses, a maturation defect of the RTC in PIC will induce an accumulation of whole viral capsids in the nuclear pore. Alternatively, the absence of the triplex DNA in the cPPT mutant linear DNA will prohibit interaction with transport proteins by prohibiting translocation of the HIV-1 genome by the nuclear pore. In both cases, the cPPT mutant virus DNA accumulates as linear DNA in the vicinity of the nuclear membrane. Figure 7 shows the results of transduction experiments using human cord blood CD34 + cells. (A) FACS analyzes of human cord blood CD34 + cells transduced for 24 hours in the presence of 100 ng / ml viral P24 under conditions described herein. Analysis was performed 48 hours after washing the cells at the end of the 24 hour transduction period. The percentages are expressed as proportions of hematopoietic cells morphologically controlled. Mean X indicates the mean value of green fluorescence intensity. (B) GFP expression was analyzed on day 5 post transduction. Figure 8 diagrammatically depicts the viral dosing effect on transfection efficiency. (A) The percentage of CD34 + cells expressing eGFP. (B) The mean value of GFP fluorescence intensity of CD34 + eGFP + cells. (C) The mean value of GFP fluorescence intensity of CD34 + eGFP + cells multiplied by the percentage of CD34 + eGFP + cells. CD34 + cells were transduced for 24 hours under the conditions specified herein with the lentiviral vector 10 including the eGFP coding sequence under CMV promoter control, and including (thick line) or without (dashed line) the triplex structure. Analysis was performed 48 hours after washing the cells at the end of the 24 hour transduction period. Figure 9 depicts FACS analysis of human cord blood CD34 + cells transduced for 24 hours under the conditions described in the text with a lentiviral vector having an intact HIV-1 LTR (left panels) or a U3-deleted HIV-1 LTR (right panels) and an internal CMV promoter (upper panels) or an EF-1 alpha (lower panels) promoter. (A) Analysis was performed 48 hours after washing the cells at the end of the 24 hour transduction period. (B) Analysis was performed 120 hours after cell lavage at the end of the 24 hour transduction period. The analysis distinguishes between luminous (immature) and dark CD34 cells.

The percentages are expressed as proportions of hematopoietic cells morphologically controlled. The second number, when indicated, represents the mean green fluorescence.

DETAILED DESCRIPTION OF THE INVENTION

We have previously shown that HIV-1 has developed a complex reverse transcription strategy that differs from that of the two-step oncoviruses that occur at the center of the genome: an additional initiation of positive strand synthesis coupled with a termination step. HIV-1 positive strand DNA is synthesized as two discrete half-genomic segments. An additional copy of the cis-active sequence of the polypurinic tract, present at the center of all the lentiviral genomes (cPPT), initiates downstream positive chain synthesis (Charneau et al., 1991, 1992). The upstream positive strand segment initiated at the 3'PPT will, after a chain transfer, proceed to the center of the genome and end after a discrete chain shift event (Figure 6A). This last reverse HIV-1 reverse transcriptase event is controlled by the cis-active sequence of the central termination sequence (CTS) that ejects HIV-1 reverse transcriptase (RT) at this site in the specific context of chain shift synthesis Charneau et al., 1994; Lavigne et al., 1997). Thus, the HIV-1 reverse transcription end product is a linear DNA molecule which carries in its center a stable DNA double loop of 99 nucleotides, referred to herein as central triplex DNA (Figure 6A).

Both the central initiation and termination mutant viruses synthesize a reverse transcription product devoid of a wild-type triplex DNA. As shown in Figure 6A, in the case of a central initiation mutant, the end product is a continuous double-stranded linear DNA lacking the central triplex (Charneau et al., 1992). The downstream positive strand segment initiated in cPPT is not synthesized. Thus, no chain shift occurs at the center of the genome; the elongation of the transferred positive strand proceeds throughout the genome. In the case of a central termination mutant, central chain shift events are no longer controlled by the mutated CTS sequence and longer, randomly distributed positive strand overlays are generated compared to the discrete wild type triplex DNA (Charneau et al. , 1994). Mutations in cPPT or CTS cis-active sequences severely impair HIV replication, suggesting a direct role of central triplex in the retroviral life cycle (Charneau et al., 1992; Charneau et al., 1994 ). The present invention describes that the central triplex DNA of HIV-1 is involved in a late step of nuclear import of the HIV-1 genome immediately prior to or during translocation of viral DNA through the nuclear pore. For this reason the distinctive features of lentiviral reverse transcription are responsible, at least in part, for the unique ability of lentiviruses, among retroviruses, to replicate in non-dividing cells. The invention also discloses that poor HIV gene transfer vectors in central triplex DNA exhibit a strong nuclear import defect. This invention provides that insertion of the central cis-active sequences of the HIV-1 genome into a previously described HIV vector (Naldini et al., 1996) enhances transduction efficiency by complementing the nuclear import defect of the original DNA vector at levels of the wild type. This finding provides additional, independent evidence of the role of central triplex DNA in HIV-1 nuclear import. The description of triplex DNA as a determinant of nuclear import of lentiviruses has important implications for the design of efficient lentiviral vectors. Since the infection of non-dividing target cells by lentivirus relies on its use of an active nuclear import pathway, it is preferable to maintain the lentiviral nuclear import determinants on derived vector elements. Classical retroviral vector elements are substitution vectors in which the complete viral coding sequences between the LTRs and substituted by the sequences of interest are deleted (Miller AD, 1997). In the case of lentiviral vectors, this classical strategy leads to the deletion of the cPPT and CTS central cis-active sequences. The important role of triplex in nuclear HIV importation implies that such vector substitution elements are not optimal. This finding establishes that the nuclear import determinant of triplex DNA is operative in the heterologous context of a lentiviral vector derived from HIV-1. Triplex DNA per se outside the context of the native HIV-1 genome can promote the nuclear import of 13 heterologous DNA sequences (French PCT No. 9805197, April 24, 1998). The presence of the triplex DNA sequence induces a marked increase in gene transduction efficiency in hematopoietic stem cells.

Thus, in one aspect, the present invention provides nucleic acids (DNA or RNA, or analogues thereof) that are capable of participating in triplex nucleic acid (i.e., nucleic acid triple chain) structures. In embodiments of the invention, the nucleic acids are lentiviral sequences. In embodiments, nucleic acids are derived from lentiviral sequences, such as by site-directed mutagenesis (i.e., intentional deletion, insertion, or substitution of at least one nucleotide) or selective pressure mutagenesis. The nucleic acids of the invention allow high-efficiency transfer of nucleic acids into host cells, and especially into host cell nuclei. Because of this ability, genes that are operably or physically linked to the nucleic acids of the invention can be expressed at high levels and / or in a high percentage of cells exposed to nucleic acids. DNA inserted into a triple-stranded region of a lentiviral genome according to the present invention is particularly stable in the element, and helps achieve a high level of transduction and transfection.

In a preferred embodiment, the invention provides a three-stranded DNA sequence induced by the cPPT and CTS regions of a lentivirus, which is capable of inducing a high DNA vector entry rate in a host cell nucleus, or capable of increasing the nuclear import rate of vector DNA. In embodiments, the triple stranded DNA sequence may be covalently linked to a heterologous nucleic acid sequence, such as a reporter gene or gene of another interest. For example, the nucleic acid of the invention may comprise a heterologous nucleic acid sequence encoding a peptide, polypeptide, or protein. In embodiments 14, the heterologous nucleic acid sequence is present in the triple-helix region. In embodiments, the heterologous nucleic acid sequence is present in the same nucleic acid as the triple-helix region, but outside the triple helix. In embodiments, a single nucleic acid comprises more than one triple chain sequence induced by the cPPT and CTS regions of a lentivirus.

In another aspect, the present invention provides vectors such as vectors, expression vectors, integration vectors, transposons, retrotransposons, and the like, which contain at least one sequence capable of participating in the formation of triplex nucleic acid structures.

In embodiments, the vector comprises a unique sequence capable of participating in the formation of triplex nucleic acid structures. In embodiments, the vector comprises more than one sequence capable of participating in the formation of triplex nucleic acid structures. Vectors can be used to transfer the nucleic acids of the invention from one cell to another, assist in generating large amounts of the nucleic acids of the invention, and serve as a base molecule in which other nucleic acid sequences of interest (eg, genes reporter, therapeutic genes) can be inserted.

In yet another aspect, the present invention provides a method for efficiently infecting, transfecting, or transducing cells with a viral nucleic acid sequence comprising at least one sequence that can participate in triplex nucleic acid structures. In embodiments, the method comprises exposing a cell, or a plurality of cells, to the at least one copy of a nucleic acid, vector, virus, or cell of the invention. In embodiments, the step of exposing the cell or cells to the nucleic acid, vector, virus, or cell of the invention is conducted under conditions such that the nucleic acid, vector, virus, or cell of the invention can enter the cell or cells target (host). In preferred embodiments, the conditions are such that high levels of the nucleic acid, vector, virus, or cell of the invention enter the target cell or cells.

In embodiments, the method is highly efficient, allowing insertion of the nucleic acid of the invention into the nucleus of 30% or more of the cells exposed to the nucleic acid. In embodiments, the percentage of nuclei incorporating the nucleic acid of the invention is 40% or greater, for example 50% or more, 60% or more, 70% or more, 80% or more, or 90% -100% . In preferred embodiments, the percentage of nuclei incorporating the nucleic acid of the invention is greater than 80%, more preferably greater than 85%, and more preferably greater than 90%, greater than 95%. In embodiments, the cells are infected or transfected with whole viruses (including phages) or bacteria containing the nucleic acids of the invention. In embodiments, the nucleic acids of the invention are introduced into the cells using mechanical and / or chemical means (e.g., electroporation, liposome fusion). In embodiments, naked nucleic acid according to the invention is incorporated by the target host cells.

Thus, in one embodiment, the invention provides the use of a nucleotide sequence comprising the cPPT and CTS regions, which adopt a triple-stranded DNA structure (triplex) after reverse transcription, in a lentiviral or retrotransposon vector and that stimulates the entry of, and the nuclear import rate of, the DNA vector in the nucleus of a transduced cell.

In one aspect of the invention, recombinant cells containing the nucleic acids or vectors of the invention are provided. Recombinant cells may be any cell (prokaryotic or eukaryotic), including progeny or derivatives thereof. Thus, the invention includes cells which are made using a cell of the invention. In modes of execution, the cells are eukaryotic. In embodiments, the cells are mammalian cells, such as HeLa cells or hematopoietic cells, such as hematopoietic stem cells. Thus, in embodiments, the invention provides a method of transducing eukaryotic cells, wherein the method comprises using a triple-stranded DNA sequence induced by the cPPT and CTS regions of a lentivirus, which is capable of inducing a high rate of entry of DNA vector into a host cell nucleus, or capable of increasing the nuclear import rate of the DNA vector.

Because the recombinant cells of the invention can express a heterologous gene of interest at high levels, these cells can be used in numerous applications. Applications include, but are not limited to, the production of high levels of proteins of interest (such as proteins of therapeutic value) in cell culture.

In embodiments, this aspect of the invention provides a method of producing a recombinant protein of interest by exposing a target cell to a nucleic acid, vector, or virus of the invention and allowing the target cell to incorporate nucleic acid of the invention, e.g. transduction, transformation, or transfection. Upon introduction of the nucleic acid into the target cell, the cell is cultured under conditions by means of which the recombinant protein of interest is expressed, and thus produced by the target cell which is now considered a recombinant cell according to the invention. While the method may be practiced in a single individual cell, it is clear that the method will often be more practical if practiced on a collection of cells in which all cells are clones. As used herein, &quot; cell &quot; refers to an individual cell or a collection of identical cells. The method may further include purifying or isolating the protein of interest from the recombinant cell or cell culture fluid. In such a method, methods of expression and purification of proteins known to those skilled in the art may be applied. These procedures are well known to those skilled in the art and therefore need not be detailed herein.

In a preferred embodiment, this aspect of the invention provides a process for expressing a gene of interest in vitro, wherein the method comprises: a) exposing target cells to an isolated or purified nucleic acid comprising a gene of interest and at least one copy of the cis cPPT and CTS acting regions of a retrovirus, wherein the cPPT and CTS regions induce a triple stranded DNA structure, under conditions allowing nucleic acid incorporation into the target cell to create a recombinant cell, and b) culturing the cell under conditions that allow at least which part of the nucleic acid is transferred to the nucleus of the recombinant cell and the gene of interest is expressed. In embodiments, the process uses a vector according to the invention. Thus, the invention provides a method of expressing a gene of interest in vitro, for example, in tissue culture. In embodiments, the method comprises exposing target cells to a nucleic acid, vector, virus, or cell of the invention under conditions where the target cell can incorporate the molecule of the invention containing the gene of interest. The recombinant cell thus produced is then allowed to grow and replicate under conditions where the gene of interest is expressed. In embodiments, the in vitro method of gene expression is coupled to a method of purifying or isolating the protein of interest. In such embodiments, the protein of interest can be purified or isolated from other cellular components using techniques known to the skilled man, including, but not limited to, liquid chromatography, precipitation, centrifugation, gel filtration, and affinity chromatography. Appropriate techniques are known to those skilled in the art and need not be detailed herein. The invention is also directed to a recombinant cell of the invention for use in the expression of a gene of interest, for example in a subject in need of the protein expressed by the gene. Said recombinant cell may be produced out of the subject by exposure of a host cell to a vector, virus, or cell of the invention. In preferred embodiments, the recombinant cell is a hematopoietic stem cell. In embodiments, the recombinant cells may also be first purified or isolated from non-recombinant cells prior to administration.

In other embodiments, the subject is exposed to a vector, and / or virus of the invention, in an amount and form sufficient to result in the expression of the gene of interest in the subject's body. Expression of the gene of interest is preferably obtained in a target cell or tissue.

In addition, the invention also relates to an expression HIV-1 vector comprising: a) an isolated or purified nucleic acid comprising at least one copy of the cPPT and CTS cis-action regions of a retrovirus, wherein said cPPT and CTS induce a triple stranded DNA structure and b) a heterologous nucleic acid sequence, wherein the HIV-1 LTR is deleted from the promoter and the U3 enhancer, for use in the treatment of an individual suffering from or having a a high probability of developing a genetically based disease or disorder wherein said retroviral vector is in an amount sufficient to result in the expression of said therapeutic protein in an amount sufficient to treat said disease or disorder. Expression of the HIV-1 vector is used in a prophylactic, improvement, or curative treatment. The treatment may encompass a disease or blood disorder, a disease or disorder of the brain or nervous system, or a developmental disorder or disorder. Techniques for introducing and / or expressing genes in vivo are known to the person skilled in the art. The physician may select the most appropriate technique for the given protein or tissue or target cell.

Accordingly, the invention also relates to an expression vector according to the invention, which is capable of inducing a high rate of DNA vector entry into a nucleus of a host cell, or capable of increasing the vector import rate of DNA, to treat a host.

According to the prior aspects of the invention, a kit is also provided. The kit may contain at least one nucleic acid, at least one vector, at least one virus, or at least one cell of the invention, or a combination of any or all of the foregoing. The kit can provide each of the above exemplary embodiments of the invention together in a single composition or separately, as for example in different containers. In embodiments, the kit includes some or all of the reagents and provisos required to use the nucleic acids, vectors, viruses, and cells of the invention for the desired purpose. The present invention discloses an original nuclear import mechanism of HIV-1 with a crucial role of a triple-stranded DNA structure, the triplex DNA, in this mechanism. HIV-1 has developed a complex reverse transcription strategy whereby a central chain shift event following central reverse transcription initiation and termination creates a triplex DNA at the center of non-integrated linear HIV-1 DNA molecules. This triplex DNA acts in turn as a cis-active determinant of the nuclear import of the HIV-1 genome. The invention shows that central initiation and termination, two distinct steps of reverse transcription of HIV-1, are responsible for the ability of HIV-1 to infect undifferentiated target cells.

The examples of the invention further show that the lack of the triplex DNA leads to a virus that is almost non-infectious in dividing or dividing cells. Although mutations in cPPT do not affect the viral DNA synthesis rate or their ability to integrate in vitro, most of the retrotranscribed DNA molecules of the cPPT mutant virus accumulate over time as non-integrated linear DNA. Instead, the wild-type virus linear DNA is almost completely processed into integrated provirus and circular DNA. Intracellular DNA profiles of cPPT mutant viruses point to a nuclear import defect, viral DNA accumulates as linear molecules as a consequence of its lack of access to the nuclear compartment where it could integrate or circularize. A late import defect of viral DNA, most likely affecting translocation through the NPC, is demonstrated by fractionation of infected cells and direct visualization (FISH) of the intracellular viral DNA. Triplex defective linear DNA molecules associate with the nuclear membrane. The invention focuses on the analysis of mutant cPPT viruses, which are characterized by the absence of a central triplex DNA. Most of the experiments presented herein were also conducted on mutant CTS viruses previously described (Charneau et al., 1994), with the same results (data not shown). In the CTS mutant virus, reverse transcription produces linear DNA molecules containing larger, randomly distributed positive overlays, compared to the discrete central triplex DNA of the wild type virus. Thus, not only the presence of triplex DNA, but also its structural integrity, is important for the nuclear import of HIV DNA. The invention shows that the triplex DNA is operative in the context of a vector system based on HIV-1. Their insertion into a vector devoid of triplex reverses a strong nuclear import defect of the DNA vector to wild-type nuclear import levels. The central triplex DNA is a common nuclear import determinant of lentivirus. The location of the central triplex DNA was precisely defined in the case of HIV-1. The center chain shift begins at the first nucleotide following the cPPT sequence (Charneau and Clavel, 1991) and generally ends 99 nucleotides downstream at the ter2 site of the CTS sequence (Charneau et al., 1994). The three-dimensional configuration of the three DNA strands of the triplex is unknown. Nevertheless, the presence of a triplex DNA at the center of the genome can be generalized to all lentiviruses. A central copy of PPT is a common feature of all lentiviral genomes and a putative CTS terminator, revealed by the presence of (A) ne (T) n tracts, also exists approximately 100 nucleotides downstream (Charneau et al., 1994 ). The central triplex DNA of the ungulate lentivirus EIAV was recently characterized (Stetor et al., 1999). A central chain discontinuity in VISNA virus DNA, referred to as an aperture, but more likely a cut resulting from the central chain shift, was revealed by SI nuclease cleavage (Harris et al., 1981). Since mitotic independent replication has been described for most lentiviruses (Gartner et al., 1986; Thormar, 1963), the role of triplex DNA in the nuclear import described herein for HIV-1 can be generalized to all lentiviruses .

Without being limiting, the invention provides a mechanism hypothesis for the role of central triplex DNA in HIV-1 nuclear import as follows: A triple-stranded DNA structure acting as a cis-determinant of its nuclear import is a new biological phenomenon without known cellular or viral counterparts. Any hypothesis of a molecular mechanism that describes the role of central triplex DNA in nuclear HIV import is then speculative. The central triplex may act as a viral determinant for initiation of HIV DNA filament uptake by the nuclear pore. This may be achieved by direct interaction of the triplex DNA with pore components or alternatively by interaction of the triplex with cellular or viral proteins that move between the cytoplasm and the host cell nucleus and may carry the HIV genome to the nucleus. Translocation of the 9.7 kb HIV genome through a nuclear pore of maximal diameter of 26nm should occur in a specific orientation after recognition of one end of the HIV-1 DNA strand to initiate uptake. A similar situation arises in the nuclear export of messenger RNA, where the uptake of the RNA filament through the pore is guided by the 5'Cap structure (Hamm and Mattaj, 1990).

Although the conformation of HIV-1 PICs is not well known, it has been established that the binding of the ends of the linear DNA are occurring together, probably after dimerization of the integrase proteins bound in the tops of the LTRs (Miller et al., 1997). Interestingly, the cis-active cPPT and CTS sequences are found centrally in all lentiviral genomes. A logical structure for lentiviral PICs would be a double strand DNA, symmetrically folded on both sides of the central triplex by integrase dimerization (Figure 6B). The triplex will then constitute a vertex of a filamentous HIV-1 PIC and the integrase dimer the opposite vertex. In cPPT mutant PICs, the absence of a triplex DNA would lead to their lack of recognition by the nuclear pore machinery or the coming proteins. The identification of the ligand proteins of central triplex DNA promises to be of primary importance both for our understanding of the nuclear import of HIV-1 PIC and for the eventual development of drugs marking this step of HIV replication. 23

Another hypothesis of mechanism to explain the properties of triplex mutant viruses would involve a defect in HIV capsid maturation in PICs prior to translocation of viral DNA into the nucleus. According to this model, defective viral DNA in triplex would remain incorporated as integral viral capsids, incapable of translocation. Retroviral reverse transcription does not occur at high dilution of the viral components in the cytoplasm of infected cells, but requires the structural environment of a reverse transcription complex where the components are limited in a set of capsid proteins. The size of the HIV capsid exceeds the maximum exclusion diameter of a nuclear pore (Dworetzky et al., 1998; Gelderblom, 1991). Thus, before viral DNA can enter the nucleus, HIV reverse transcription complexes must undergo maturation in PICs of size compatible with translocation through nuclear pores (Karageorgos et al., 1993). The maturation of viral capsids prior to nuclear translocation is well established in other viral systems in which the replication cycle involves translocation of the genome DNA through the host cell nuclear membrane (Whittaker and Helenius, 1998). While reverse transcription within viral capsids has been physically demonstrated for MLV (Bowerman et al., 1989), this has not yet been possible for HIV-1 due to possibly the fragility of HIV-1 capsids (Borroto-Esoda and Boone , 1991). The HIV reverse transcription complex contains numerous copies of the RT polymerase (approximately 30 to 50 per capsid) (Panet et al., 1975). Due to the important distribution of HIV-1 reverse transcriptase, high enzyme stoichiometry for the viral RNA model is required to overcome a number of reverse transcription limiting steps such as chain transfer or polymerization pauses during positive strand synthesis and negative and accurate formation of the central triplex (Klarmann et al., 1993; Charneau et al., 1994). this strongly suggests that the central terminus, the last event of the lentiviral reverse transcription, occurs in an integral capsid structure. The central terminus, which marks the end of viral DNA synthesis, may be a necessary signal for the clearance of viral capsid DNA and its subsequent translocation into the nucleus.

These two putative molecular mechanisms for HIV-1 triplex DNA-mediated nuclear import are not mutually exclusive. The formation of a central triplex DNA may activate the maturation of viral capsids in PICs, thereby rendering the triplex DNA accessible to viral proteins. The fact that integrity of the central triplex DNA is required for entry of the HIV-1 genome into the host cell nucleus implies that the entire DNA synthesis process, including the last central chain shift event, is completed prior to translocation of the PIC of HIV through the nuclear pore. The subcellular distribution of lentiviral reverse transcription is currently under debate and whether HIV replication occurs in a specific cell compartment is still an open question. Based on fractionation studies, it has been reported that reverse transcription of HIV can occur completely in the cell nucleus (Bukrinsky et al., 1993a). However, fractionation techniques do not distinguish between an intranuclear location and association with the nuclear membrane. Other authors have proposed, on the contrary, that the association of the reverse transcription complex with the cytoskeleton is a prerequisite for viral DNA synthesis (Bukrinskaya et al., 1998). Kinetic studies of HIV-1 DNA synthesis and its association with the nuclear fraction indicate that the latter process is much faster than the first. DNA synthesis of HIV-1 in the course of a single reverse transcription cycle only reaches a plateau 24 to 48 hours post-infection, whereas more than 95% of HIV-1 DNA fractionates with nuclei of infected cells as as early as 4 to 6 hours after infection (Barbosa et al., 1994). We therefore favor a third possibility that lentiviral reverse transcription takes place primarily in the immediate vicinity of the nuclear membrane and NPCs within the viral capsid, although additional experiments are required to confirm this hypothesis. Cell mitosis does not provide an alternative route for entry of defective triplex viral DNA into the nucleus of host cells. The present Examples show that central triplex mutant viruses are heavily hindered in their replicability not only on dividing but also dividing target cells. Conversely, insertion of a triplex DNA sequence into an HIV-1 based vector stimulates gene transduction in both split and non-split cells. This differs from the published phenotype of mutant MA / Vpr viruses, where a replication defect was described exclusively in non-dividing cells (Bukrinsky et al., 1993b; Heinzinger et al., 1994; von Schwedler et al., 1994). Thus, MA / Vpr mutants would behave as mitotic-dependent oncoviruses. One possible explanation for the different behavior of central triplex mutants is that these viruses are defective in a late step of the nuclear import process, therefore the defective triplex defective DNA molecules associate with the nuclear membrane. This intimate association persists during mitosis. Mutant viral DNA can be incorporated into mitotic nuclear membrane vesicles, where it can not reach the cellular chromosomes, as reported in the case of NLS-LacZ proteins (Bonnerot et al., 1987). Mutations in cellular NLS, which inhibit interactions with karyopherins, however, are known to induce cytoplasmic accumulation of the mutated protein (Kalderon et al., 1984; Lanford and Butel, 1984). Thus, mutations in the NLS sequences contribute to the caryophilic properties of HIV-1 PICs should induce cytoplasmic retention of viral DNA, as previously suggested (Gulizia et al., 1994). It is then possible that mutant MA / Vpr virus PICs reach the cell chromosomes after disruption of the nuclear membrane during mitosis. However, there is still no direct experimental evidence of a nuclear import pathway dependent on mitosis of lentiviral genome DNA. As the published phenotype of mutant MA / Vpr viruses should be viewed with some caution, the same caution should be applied to the initial hypothesis that a lentivirus nuclear import deficiency should lead to a replication defect exclusively in non-dividing cells. Whether lentiviruses can adopt a mitotic-dependent nuclear import strategy, or whether the active nuclear import of lentiviral genomes occurs in both split and non-split cells, remains an open question. The present invention provides drawings for lentiviral vectors. Since target cell infection without lentivirus division is based on its use of an active nuclear import route, it is important to maintain the lentiviral nuclear import determinants on derived vector elements. Classical retroviral vector elements are substitution vectors in which the entire viral coding sequences between the LTRs are deleted and replaced by the sequences of interest. In the case of lentiviral vectors, this classical strategy leads to deletion of the cPPT and CTS central cis-active sequences. The important role of triplex in nuclear HIV importation implies that such vector substitution elements are not optimal. Thus, insertion of the HIV triplex DNA into the HR GFP substitution vector (Naldini et al., 1996) increased its gene transduction efficiency by complementation of a nuclear import defect of the HR vector genome at an import rate of DNA near that of wild type HIV-1. It should be noted that whereas HIV vectors lacking a triplex DNA are still capable of gene transduction (Figure 2C, 2D), the residual replication of mutated viruses in cPPT is absent or extremely low (Figure 6A, 6B) . One possible explanation is that triplex-independent nuclear import can occur in the case of a small vector genome (approximately 4 kb for HR-GFP), but the presence of a triplex DNA will be required for the import of the native HIV-1 genome of 9.7 kb. In fact, the relatively ineffective active nuclear import of DNA molecules as large as 3 to 4 kb was reported (Hagstrom et al., 1997).

The cis-active sequences responsible for formation of the triplex DNA are at the center of all lentiviral genomes. This central position was developed because of its structural implications for PIC conformation in that symmetric folds of the left and right arms of the linear DNA molecule around the triplex could be required for its efficient uptake through NPCs ( Figure 7B). If this is true for the virus, then a central position of the triplex DNA may also be required for effective nuclear import of vector genomes.

EXAMPLES The invention will be further clarified by the following examples, which are intended purely as examples of the invention.

Example 1: Experimental Procedures. Cells

MT4 cells are HTLV-1 transformed human CD4 + T cells that allow acute cytopathic HIV-1 infection (Harada et al., 1985). H9 cells are less permissive to HIV but allow chronic production after infection. MT4 and H9 cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS). Peripheral blood lymphocytes (PBLs) were obtained from healthy donors, stimulated with æg / ml phytohemagglutinin (Wellcome), and maintained in the presence of Interleukin-2 (10% lymphocult;

Biotest Diagnostics). 293T cells were grown in DMEM medium supplemented with 10% FCS. P4 indicator cells are infectious CD4 + HIV HeLa cells carrying the LacZ gene under control of HIV-1 LTR (Charneau et al., 1994). P4 cells are grown in DMEM medium supplemented with 10% FCS and 500pg / ml G418.

Collection and fractionation of cells.

Cord blood samples were collected with the consent of the mothers. CD34 + cells were purified as previously described (Robin, C. et al., 1999) using the miniMACS immunomagnetic droplet separation system (Milteny Biotec). The purity of CD34 + cells separated by droplets was greater than 75%. CD34 + CD3810 fractions were further purified by cell selection with a FACS Vantage ™ equipped with an ionized argon laser (Becton Dickinson) using murine monoclonal antibodies (MoAbs) directed against CD34-PE-Cy5 (Immunotech) and CD38-PE (Becton Dickinson). CD34 + cells were either frozen in fetal calf serum (FCS, Stem Cell) containing 10% DMSO (Sigma) or used immediately.

DNA elements

Proviral Plasmids:

Targeted mutagenesis was performed as previously described (Kunkel, 1985) on M13mpl8 carrying a 1.1 kb EcoRI insert (4684 to 5779) of the infectious molecular clone pLAI3. Mutagenic primers were as follows: cPPT-AG 5'pCAATTTTAAAAGAAGAGGGGGGATT 3 '(SEQ ID NO: 1) cPPT-D: 5'pATTCATCCACAACTTCAAGCGCCGCGGTGGTATTGGGGGGTAC 3' (SEQ ID NO: 2). The pcPPT-AG, pcPPT-D, pcPPT-25 and pCTS were constructed by retro-cloning the mutated EcoRI fragment in pLAI3. 29

Plasmid vectors:

Plasmid vectors were derived from HR'CMVLacZ (Naldini et al., 1996). The LacZ reporter gene was replaced by the EGFP gene (Clontech). The EGFP gene was amplified by PCR using Pfu (Stratagene) polymerase from plasmid pEGFP-N1, adding restriction sites BamHI and XhoI at the 5 'and 3' ends respectively. The PCR primers were as follows:

Bam GFP: 5'CC GGATCC CCA CCG GTC GCC ACC 3 '(SEQ ID NO: 3)

Xho GFP: 5'CC CTCGAG CTA GAG TCG CGG CCG 3 '(SEQ ID NO: 4). The HR GFP vector was constructed by retro-cloning this PCR fragment at the BamHI and Xhol sites of pHR'CMVLacZ, substituting the LacZ ORF with EGFP. TRIP AU3 CMV GFP and TRIP AU3 PL CMV GFP:

First, a subclone containing a single LTR was constructed and designated pUCLTR. The KpnI / XbaI fragment of TRIP GFP spanning its 3'LTR was cloned into pUC18. Next the EcoRI site was destroyed by padding, creating the vector pUC LTR RI-. Divergent PCR was performed on pUC LTR RI- with the aim of amplifying the entire plasmid with the exception of the promoter and the U3 sequence enhancer. The primers were: DU3 -: 5'CGGAATTCGGATCCGCGGCCGCATCGATCTTGTCTTCGTTGGGAGTG 3 '(SEQ ID NO: 5)

DU3 +: 5'CGGAATTCAGCCGTCTCGAGAGATGCTGCATATAAGCAGC 3 '(SEQ ID NO: 6).

The primers contain the restriction sites to be inserted instead of the U3 sequence including the EcoRI site 30 present in each primer. The PCR product was digested with EcoRI and then used to transform competent bacteria. The plasmid thus constructed was designated pLTR AU3 RI-. The inserted polylinker instead of the U3 sequence in pLTR AU3 RI-is:

Cyan-NotI-BamHI-EcoRI-Mulo-XhoI. Plasmid TRIPAU3 PL GFP was constructed by replacing the KpnI / Nhel fragment of GFP TRIP containing the 3'LTR with the KpnI / XbaI fragment of pLTR AU3 RI- (the Nhel and XbaI restriction products are compatible). Then the polylinker (PL) was deleted from pLTR Δυ3 RI- by digestion with Clal / XhoI and filling. The TRIP plasmid AU3 GFP was constructed by replacing the KpnI / Nhel fragment of TRIP GFP with the KpnI / XbaI fragment of pLTR AU3 APL RI-.

A 17 8 bp fragment of pLAI3 (4793 to 4971) spanning cPPT and CTS was amplified by PCR. 5 'NarI restriction sites were added from the primers with the aim of inserting this fragment into the single Ciai site of HR GFP:

Nar TRIP +: 5'GTC GTC GGCGCC GAATTC ACA AAT GGC AGT ATT CAT CC 3 '(SEQ ID NO: 7)

Nar TRIP-: 5'GTC GTC GGCGCC CCA AAG TGG ATC TCT GCT GTC C 3 '(SEQ ID NO: 8) Insertion of this triplex sequence in the correct orientation gave rise to the TRIP GFP plasmid vector, and a TRIPinv GFP in the reverse orientation. Alternatively, the same triplex fragment was amplified from the plasmids pcPPT-AG, pcPPT-D, pcPPT-225, and pCTS to generate vectors including the same mutations in cPPT or CTS as the corresponding viruses. TRIP EFla GFP and TRIP ÀU3 EFla GFP: The TRIP GFP CMV promoter was replaced by the EFla promoter. The triplex sequence and the EFla promoter were first separately amplified, with primers overlapping. The triplex sequence was amplified with the primers Nar TRIP + and Mlu TRIP- in the pLai matrix and the EFla promoter was amplified in the pEFpgkneo template with primers Mlu EF1 + and Bam EF1,

Nar TRIP +: 5'GTC GTC GGCGCC GAATTC ACA AAT GGC AGT ATT CAT CC 3 '(SEQ ID NO: 9)

MluTRIP-: 5'AGC CTC ACG ACGCGT AT CAG CCA AAG TGG ATC TCT GCT G3 '(SEQ ID NO: 10)

MluEFl +: 5'-CTG AT ACGCGT CGT GAG GCT CCG GTG 3 '(SEQ ID NO: 11)

Bam EF1 -: 5'CG GGATCC TGT GTT CTG GCG GCA AAC3 '(SEQ ID NO: 12)

Thereafter, a second round of PCR was run on a mixture of the first two PCR products, using the outer primers Nar TRIP + and BamEF1-. The triplex sequence and EFla promoter were pooled by this technique. The TRIP EFla GFP plasmid was constructed by substituting the EcoRI / BamHI fragment of TRIP GFP containing the triplex sequence and the CMV promoter by the EcoRI / BamHI-digested TRIP PCR product EFla (Nar TRIP + primer has an EcoRI site at its 5 ' ').

To construct the TRIP AU3 EFla GFP plasmid, the EFla GFP TRIP EcoRI / BamHI fragment containing the triplex sequence and the EFla promoter was inserted into TRIP AU3 GFP instead of the EcoR1 / BamHI fragment containing the triplex sequence and the CMV promoter.

Virus and vector production

For the investigations illustrated in Figures 1-6, viruses were produced by transient transfection of HeLa cells by the calcium phosphate co-precipitation technique. The vector particles were produced by transient co-transfection of 293T by the plasmid vector, and encapsidation plasmid (p8.2) and a VSV envelope expression plasmid (pHCMV-G, (Yee et al., 1994)) as previously described (Naldini et al., 1996). All virus and vector supernatants were treated with DNasel (1æg / ml in the presence of

MgCl 2 ΙμΜ) for 15 minutes at 37 ° C.

Production of lentiviral vector particles

For the investigations shown in Figures 7-9, viruses were produced by transient co-transfection of 293T by the plasmid vector, an encapsidation plasmid (p8.2) and a VSV envelope expression plasmid (pHCMV-G, (Yee et al. ., 1994)), as previously described (Naldini et al., 1996).

All virus and vector supernatants were treated with DNasel (1æg / ml in the presence of MgCl 2 ΙμΜ) for 15 minutes at 37øC.

Virus and Vector Titrations

For the investigations shown in Figures 1-6, a virus titration cycle was performed in triplicate by infection of plated P4 cells in 96-well plates with equivalent amounts of particles (Âμg of p24 viral antigen per well) in the presence of DEAE-dextran 20 μΜ. The Saquinavir protease inhibitor (Roche) was added (ΙμΜ) throughout the experiment to restrict the analysis to a single cycle of infection. Cellular mitosis was inhibited by treatment with antifungal agent (8μΜ), on the day prior to infection. Β-Galactosidase activity was measured 48 hours post-infection using a β-Gal-chelmuminescent reporter gene assay (Boehringer). HeLa cells were infected in triplicate with equivalent amounts of vector particles (5ng P24 per well). At 48 hours post-transduction, the medium was replaced with 200μl of TNB (50mM Tris pH 7.5, 150mM NaCl) and the live cell fluorescence was quantified using a microplate fluorometer (Victor2, Wallac) and filters adapted to EGFP (excitation: 485 nm, emission: 520 nm).

Transduction protocol

For the investigations disclosed in Figures 7-9, 24- or 96-well tissue culture plates were coated with fibronectin (Bio-Whittaker Europe) according to the manufacturer's instructions. Human CD34 + populations were plated immediately after purification or thawing at 2 to 3 X 10 5 cells / ml in serum free medium (IMDM containing 11.5 μg æ-thioglycerol, 1.5% BSA (both from Sigma), sonicated lipids and iron-saturated human transferrin) or a-MEM containing 10% FCS in the presence of 4pg / ml polybrene (Sigma) and 4 recombinant human (hu) cytokines (r): Staphylococcal Cell Factor (SCF, 100ng / ml, provided by Amgen), Ligand-Flt3 (FL, 100ng / ml, Diaclone), IL-3

(60ng / ml, Sandoz), and Pegylated Megacarocyte-Rhu (PEG) (MGDF) Growth Factor and Differentiation (1 ng / ml, Amgen), and lentiviral viruses concentrated at 100 ng viral P24 / ml for 24 hours. The cells were then washed and cultured under lympho-myeloid conditions (on culture tissue culture plates precoated with MS5 cells in RPMI supplemented with 10% human serum, 5% FCS and the following 7 cytokines: rhu-SCF (50 ng / ml), rhu-IL-2 (5 ng / ml), rhu-FL (50 ng / ml), rhu-FL (50 ng / ml), rhu-MGDF -IL-15 (10ng / ml), and rhu IL-7 (20ng / ml) (the three IL-

Diaclone) for 48 hours. Next, the expression of eGFP in the CD34 + cell fraction was evaluated using a CD34-PE-Cy5 MoAb (Immunotech). The analysis was performed on a FACS Scan using Cellquest software (Becton Dickinson).

Clonogenic assays and long-term culture (LTC)

Clonogenic progenitors of fresh human CB cells were analyzed in 30% FCS containing 0.8% methylcellulose, 1% deionized BSA, and 10-4M 2-mercaptoethanol in the presence of rhu-SCF 50ng / ml, rhu-GCSF 10ng / ml (Amgen), rhu-IL3 2ng / ml, and rhu-2U / ml EPO (Amersham). NOD-SCID bone marrow (BM) cells grafted from mice were plated in the presence of rhu-SCF, -IL-3, -EPO, and -GM-CSF (10 ng / ml) as described (Pflumio, F. et al. al., 1996). The progenitors were labeled on day 14-16 according to criteria already described (Croisille L. et al., 1994) and EGFP expression observed by fluorescence microscopy using a Nikon Eclipse TE300 microscope. Long-Term Culture (LTC) was performed as previously described (Issaad C. et al., 1993) or limit dilution in 96-well plates using the FACS advantage equipped with an ACDU (BD) or bulk culture in 24-well plates containing a confluent layer of murine MS5 stromal cell line. After 5 to 10 weeks, the adherent and non-adherent cells were harvested and plated for the clonogenic assay. For both the clonogenic and CTL-IC assays, colonies were selected individually and frozen prior to PCR analysis.

PCR analysis

PCR analysis was performed on genomic DNA obtained from colonies derived from clonogenic progenitors or from clones grown in lympho-myeloid cultures. Digested cells and proteins were lysed in 20μl of buffer containing proteinase K (10pg / ml), KCl (50mM), Tris-HCl (10mM, pH 8.3), MgCl2 (2.5mM), gelatin (0.1mg / ml ), NP40 (0.45%), and Tween 20 (0.45%). Genomic DNA amplification was performed with the sense primer 5'CCCTCGAGCTAGAGTCGCGGCCG 3 '(SEQ ID NO: 13) and the 5'-antisense primer 5'CCGGATCCCCACCGGTCGCCACC 3' (SEQ ID NO: 14) at the cooking temperature of 62 ° C. Amplification resulted in an 800 bp product.

Vector and viral DNA analysis P4 or MT4 cells were infected at a high multiplicity of viruses (150 ng p24 per 106 cells) or transduced by vectors (25 ng p24 per 106 cells) in the presence of 20 μg / ml DEAE- dextran in the case of P4 cells. DNA from infected or transduced cells was extracted at various times, restricted, and analyzed by Southern blot. In all cases contaminating bacterial plasmid DNA was removed from the analysis by DpnI digestion. DNA from infected cells was digested by MscI and XhoI and DNA from cells transduced by EcoNI, Avall and XhoI. After electrophoresis and transfer of 1æg of digested DNA, the membranes were hybridized with randomly primed [32 P] labeled DNA probes (Rediprime II, Amersham). The virus-specific DNA probe was amplified by PCR from the template plasmid pLAI3 using the following primers: 5Msc: 5'AGA AGA AAT GAT GAC AGC ATG 3 '(SEQ ID NO: 15) 3Msc: 5'TGC CAG TTC TAG CTC TG 3 '(SEQ ID NO: 16). The resulting 1680 bp DNA fragment (from position 1818 to 3498 of pLAI3) overlaps the MscI restriction site at position 2655 of viral genomes. The vector probe was synthesized by PCR in pTRIP GFP with primers: 5ECONI: 5'CAG GGA CTT GAA AGC GAA AG 3 '(SEQ ID NO: 17) 3EcoNI: 5'GCT TGT GTA ATT GTT AAT TTC TCT GTC 3' (SEQ ID NO: 18) The vector probe is a 1027bp fragment (from position 649 to 1676 of pTRIP GFP) and overlaps the EcoNI site at position 1156 of vector genomes.

To analyze the amount of wild type retrotranscript DNA and PPT-AG and cPPT-D viruses, a similar protocol was followed except that DNA extracted at 12 hours postinfection was restricted by MscI and DpnI. The probe used for hybridization was the 1.9 kb MscI internal fragment of pLAI3. Hybridization signals were quantified using a &quot; phosphorimager &quot; (Molecular Dynamics) and ImageQuant software.

Hybridization in si tu

P4 cells were infected at high multiplicity (2pg p24 antigen per virus per 106 cells) in the presence of 20æg / ml DEAE dextran. At 24 hours postinfection, cells were trypsinized, extensively washed (to remove adsorbed viral particles on the plasma membrane), and re-plated on glass slides in 24-well plates. Cells were grown for an additional 48 hours and fixed in 4% PFA / PBS for 20 minutes at room temperature. Cells were washed in PBS and permeabilized by 0.5% Triton / 0.5% Saponin in PBS for 5 minutes at room temperature. Dehydrated samples were treated with RNase A (200ug / ml in 2X SSC), one hour at 37øC and proteinase K (6æg / ml in PBS) for approximately 5 minutes. Samples were denatured by incubation in 70% deionized formamide / 2X SSC for 2 minutes at 70 ° C followed by 30% deionized formamide / 2X SSC for 2 minutes at 70 ° C. Hybridizations were performed overnight at 37 ° C using a cut-translational biotin-labeled plasmid pLAI3 (50% deionized formamide, 10% dextran sulfate, 10pg / ml salmon sperm DNA, 0% Tween 20, 1% in 2X SSC). The samples were extensively washed (serial wash in 2 x 37 SSC / 50% formamide at room temperature and then at 50 ° C). Detection of hybridized probes was performed using the Tyramid-Streptavidin TSA-Direct (NEN) kit according to the manufacturer's instructions.

Example 2: Central initiation of reverse transcription is an essential step of the HIV-1 replicative cycle

In a previous work, we have shown that conservative mutations in the cPPT and CTS sequences have seriously impaired virus replication (Charneau et al., 1992; Hungnes et al., 1992). A central initiating mutant virus (cPPT-225) and a terminating mutant virus (CTS) respectively showed a four fold and ten fold decrease in infectivity in a single titration experiments. In order to inactivate the cPPT function, semi-conservative mutations were introduced into the coding region of the overlap integrase. In the mutant cPPT-D virus, the shift from lysine to arginine at position 188 allowed the introduction of a total of 10 mutations in the 19 nucleotide sequence of the PPT primer (Figure 1A) (Huber and Richardson, 1990). The effect of this amino acid change in virus replication was conferred by construction of the control cPPT-AG mutant virus, in which a single purine to purine mutation, with respect to the polypurine nature of cPPT, induced the same amino acid change. The presence of a triplex DNA in the wild-type and cPPT-AG virus retrotranscript DNA, and its absence of cPPT-D virus DNA, was confirmed by DNA cleavage by Nuclease &quot; Hirt &quot; DNA. of infected cells as previously described (Harris et al., 1981; Charneau and Clavel, 1991). Viral infectivity was first evaluated in experiments of classical kinetic replication in cell culture. PHA-stimulated peripheral blood lymphocytes (PBLs) and MT4 cells were infected with equal numbers of viral particles, normalized according to the content of the capsid (p24) protein viral supernatants, and reverse transcriptase activity was followed over of the time in the culture supernatants (Figure 1B). The growth curves of the HIV-1 LAI and cPPT-AG wild type virus were similar in both cell systems. The fact that the K188R mutation occurs naturally in some HIV-1 isolates has already been suggested to have little or no effect on integrase and PPT functions. In contrast, when PBLs were infected with cPPT-D mutant viruses, no replication was detected during the 15-day culture. The same was true for MT4 cells, despite their high susceptibility to HIV infection. The cPPT-D mutant viruses also were not infectious in immortalized cell lines such as H9 or CEM (data not shown). Virus infectivity was then quantitatively analyzed by titrations based on a single replication cycle (Figure 1C). P4 (HeLa CD4 LTR-LacZ) indicator cells (Charneau et al., 1994) were infected with equivalent numbers of virus particles of the different viruses. These one-cycle titers have confirmed the near-complete loss of infectivity of cPPT-D mutant virus. In P4 cells, the infectivity of the cPPT-AG control was identical to that of the wild-type virus, whereas the infectivity of the cPPT-D mutant was strongly reduced to levels near the baseline. The same results were obtained in aphidicolin-treated P4 cells without division (Figure 1C, right panel).

These results strongly suggest that central initiation of reverse transcription is necessary for replication of HIV in cells without division as well as in proliferative cells.

Example 3: Virus production is unaffected by mutations in CPPT 39

We found that the different mutations introduced in the cPPT-AG and cPPT-D plasmid proviruses did not affect the last steps of the replicative cycle. Viral production was quantified, according to the P24 content of the supernatants, after transient transfections of HeLa cells by proviiral plasmids. The production of cPPT mutant viruses was found to be not significantly different from that of wild type viruses (Figure 2A). Consequently, the K188R mutation does not affect the last phase of HIV-1 replication. Thus, the defective step involved in the cPPT-D mutant virus phenotype must precede viral DNA expression and must belong to the early phase of the HIV replication cycle.

Example 4: cPPT mutations do not affect the reverse transcription rate of the HIV-1 genome. The effect of cPPT mutations on viral DNA synthesis was assessed by quantification of the synthesized DNA in a single reverse transcription cycle (Figure 2B). An internal MscI restriction fragment from the viral DNA of infected cells was detected by Southern blotting and quantified using the corresponding MscI DNA fragment as probe. Since the internal MscI fragment is common to the integrated proviral DNA and the non-integrated linear and circular molecules, their quantification reflects the total amount of viral DNA, regardless of its integrated or non-integrated state. To limit the analysis to the first cycle of reverse transcription, DNA from infected P4 cells was harvested 12 hours after infection, before the start of a second cycle of infection. The total amount of retrotranscribed DNA in a single reverse transcription cycle was the same after infection with the mutant cPPT-D, control cPPT-AG or wild-type virus. These experiments showed that while mutations in cPPT abolish virus replication, they do not affect the rate of DNA synthesis. The replicative defect of mutant cPPT viruses involves a subsequent step for viral DNA synthesis. 40

The ability of in vitro integration of wild-type HIV-1 and central-initiation mutant PICs was compared (Figure 2C) using the in vitro integration of HIV-1 PICs. a quantitative in vitro integration assay as described by Farnet (Farnet and Haseltine, 1990), with minor modifications. Since HIV-1 replication complexes only reside transiently in the cytoplasm of newly infected cells (Barbosa et al., 1994), the preparation of sufficient amounts of HIV-1 PICs requires massive infection and cell fractionation in 4 to 6 hours. This was achieved by co-culturing chronically infected H9 cells from wild type or cPPT-225 viruses and uninfected HUT 78 target cells. Mutant virus CPPT0225 (Figure 1A) was chosen for these experiments instead of non-infectious cPPT-D, which is not capable of establishing a chronic infection. Residual infectivity of cPPT-225 mutant virus is low but sufficient to allow it to propagate slowly in cell cultures (Charneau et al., 1992).

HIV PICs were isolated from the cytoplasm of infected cells and incubated in the presence of target DNA from the linearized Bluescript plasmid. Integration was revealed by the presence of a 12.7 kb, HIV-1 probe reactive fragment, corresponding to the expected size of the 9.7 kb linear HIV genome integrated into the 3 kb target DNA. The amount of linear DNA integrated into the plasmid DNA did not differ between the wild-type virus and the mutant cPPT 225 (Figure 2C). Thus HIV-1 PICs from the mutant cPPT retained their full ability to integrate in vitro. The defective replication step of the central triplex DNA of mutant viruses must occur after reverse transcription but prior to integration of their linear HIV genome into the host cell chromatin. 41

Example 6; Impaired nuclear import of central triplex DNA from mutant viruses

Previous experiments suggest that the replicating defect of central triplex DNA from mutant viruses was related to the access of HIV PICs to cell target chromatin. We therefore tested the hypothesis of a nuclear DNA import defect of cPPT mutant viruses. Nuclear import studies of HIV-1 PICs are hampered by the lack of a quantitative and reproducible assay for nuclear import at the viral DNA level. Once retrotranscribed into the cytoplasm, linear retroviral DNA is imported into the nucleus where it integrates or circulates. Non-integrated retroviral circular DNA, containing one or two LTRs, lies exclusively in the nucleus, and thus represents convenient markers of nuclear viral DNA import. To evaluate the nuclear import of HIV DNA, previous studies used PCR amplification of two non-integrated LTR circular DNAs. However, as disclosed herein and by Barbosa et al. (1994), because two circular LTRs represent a fraction of HIV DNA in infected cells, their detection is very sensitive to minor changes in cell physiology or virus infectivity. Thus, we designed a novel assay that allows for quantitative Southern blot monitoring of the synthesis, circularization, and integration of HIV DNA.

Briefly, DNA from infected cells is prepared at various times and digested with MscI, a restriction enzyme that cleaves the HIV-1 genome twice. Using a PCR-generated DNA probe that exactly overlaps the 5'MscI site, several specific bands are shown (Figure 3A). The 1.9 kb internal MscI fragment is common to all viral DNA species regardless of their integrated or non-integrated state and the quantification of this band indicates the total amount of viral DNA in infected cells. A 2.6 kb band 42 corresponds to the non-integrated linear HIV-5'-distal 5'MscI fragment. To minimize the transfer propensity due to the large size of specific fragments of circular DNA, the DNA is further cut with XhoI. One and two circular DNA LTRs appear in the bands 2.8 and 3.4 kb respectively. Once the DNA probe exactly overlaps the 5'MscI site, the intensity of each band is directly proportional to the amount of the corresponding viral DNA species. The amount of integrated proviral DNA is calculated by subtracting from the total amount of viral DNA the signals of non-integrated linear and circular viral DNA. A parallel quantification of the same population of infected cells was performed after a fractionation &quot; Hirt &quot; to separate non-integrated low molecular weight viral DNA from high molecular mass integrated proviral DNA. This gave rise to identical results, thus validating the subtraction calculation of a step.

As indicated by the kinetics of total viral DNA accumulation (1.9 kb internal fragment), viral DNA synthesis continued for 24-48 hours after infection, reflecting an asynchronous infection process. Total viral DNA amounts of cPPT-AG and cPPT-D mutant viruses were similar to those of wild-type HIV-1. As previously, mutations in cPPT did not influence the rate of DNA synthesis. Detectable amounts of full length non-integrated linear DNA were present in cells as early as 6 hours post infection (Figure 3B). Integrated proviruses and circular DNA were first detected 12 hours post infection. Integration and circularization followed to completion for an additional 36 hours.

At the end of an infection cycle, in the case of the wild type virus, about 55% of the viral DNA had integrated into the DNA of the host cell, about 35% had circulated in a circle of LTR, and a small fraction less than 10% remained in the form of stable non-integrated linear DNA 43 (Figure 3C). Notably, two circular DNA LTRs, although detectable 48 hours post infection, were only present in trace amounts. The cPPT-AG control virus DNA was processed in a manner very similar to wild-type virus DNA.

In the case of cPPT-D mutant viruses, a marked change in the intracellular viral DNA pattern was evident with a clear and persistent accumulation of non-integrated linear molecules. At 48 hours post infection, only very small amounts of a circular DNA LTR and integrated proviral DNA had been generated and more than 90% of the cPPT-D mutant DNA remained blocked in the non-integrated linear form (Figure 3C). A nuclear import defect is expected to decrease the proportion of nuclear viral DNA species (integrated proviruses and one and two circular LTRs) and to increase concomitantly the proportion of non-translocated linear DNA molecules. Thus, the intracellular DNA profile of cPPT-D mutant viruses strongly suggests a defect in viral DNA nuclear import.

Example 7: Linear DNA from central triplex DNA of mutant viruses accumulates in the vicinity of the nuclear membrane

To further characterize the nuclear import defect of central triplex DNA from mutant viruses, we address the questions of whether the mutated linear DNA molecules accumulate or not accumulate in a particular subcellular compartment. The nuclear import process can be divided into two main phases, conduction of the nuclear component to the nuclear membrane and its translocation by the nuclear pore complex (NPC). We first conducted a classical nucleotide / cytoplasmic fractionation of infected cells, followed by Southern blot detection of viral DNA. All viral DNA from all viruses was associated with infected P4 cell nuclei 24 hours after infection (Figure 4A), suggesting that the delivery of HIV DNA to the nuclear membrane was unaffected by the lack of a triplex DNA central.

To confirm that the deficient central triplex DNA molecules accumulate in the nuclear membrane, we used in situ fluorescent hybridization (FISH) to directly visualize the intracellular localization of HIV genomes. P4 cells were infected at a high multiplicity in a cycle of conditions, hybridized with a full-length HIV-1 genome probe, and observed by &quot; deconvolution &quot; microscopy. Specific stains were predominantly found in the nucleus in the case of wild-type and control cPPT-AG viruses (Figure 4B). Since FISH can not distinguish between different species of HIV DNA, these intranuclear viral DNA molecules could be either integrated proviruses or non-integrated circular DNA. Some rare genomes were associated with the nuclear membrane and probably represented the residual linear DNA detected by Southern blotting at the same time after infection. Other patches associated with the plasma membrane were most likely derived from defective particles adsorbed onto the membrane containing partially retrotranscribed genomes (Lori et al., 1992). Instead, HIV genomes were predominantly located on the nuclear membrane and almost completely absent from the nucleus in the case of cPPT-D mutant viruses. As the Southern blot DNA profile indicated that virtually all cPPT-D DNA was blocked in linear form, we can assume that these HIV genomes associated with the nuclear membrane were non-integrated linear DNA molecules. This direct visualization of viral DNA molecules in infected cells confirmed the association of the viral DNA of central triplex mutants with the nuclear membrane.

Our FISH experiments suggest that cPPT mutant viruses are defective in the translocation of their genome by NPCs. However, clear demonstration of a translocation defect by localization of mutant viral DNA on the cytoplasmic side of NPCs is not accessible through FISH experiments.

We conclude from these results, as a whole, that the central HIV-1 triplex DNA, created by central initiation and termination steps during reverse transcription, is important for HIV-1 PICs to enter the host cell nucleus. In the absence of triplex DNA, nuclear import of viral DNA is seriously impaired at one stage immediately before or during translocation of HIV-1 DNA by the nuclear pore.

Example 8: Impact of the central triplex DNA on gene transduction by an HIV-1 based vector system

Having identified central triplex DNA as a key determinant for nuclear import of HIV-1, we tested the effect of inserting the central cis-active sequences of HIV-1 into the previously described HIV-1 HR vector (Naldini et al., 1996) Figure 5A). To monitor gene transduction, a gene encoding the green fluorescent protein (GFP) has also been inserted. The vector containing a triplex DNA sequence was designated TRIP GFP. Controls included similar elements with cPPT or CTS mutated and a wild type central region inserted in reverse, nonfunctional orientation (TRIPinv GFP). The presence of a triplex DNA in retrotranscribed GFP TRIP DNA vector, and its absence of HR GFP, TRIPinv GFP, and vectors containing a mutated version of the central triplex sequence, was confirmed by cleavage with SI nuclease of Hirt DNA isolated from transduced cells. The number of vector particles produced by transient transfection was normalized prior to transduction according to capsid (p24) protein levels, reverse transcriptase activity, and the amount of RNA genomic vector in transfected cell supernatants. Similar production of the various vectors was obtained, with a linear correlation between the three normalization criteria (data not shown). Thus insertion of the central region of the HIV-1 genome into the HR vector did not influence the rate of encapsidation of genomic RNA. Hepara or non-cleaved (aphidicolin-treated) HeLa cells were transduced with equivalent numbers of HR-GFP or TRIP-GFP vector particles and GFP expression was monitored 48 hours later by quantification by fluorescence. Pseudotransduction of GFP activity due to the direct delivery of GFP proteins to target cells by the fused vector particles was calculated from the transduction of cells treated with an HIV-1 RT inhibitor (Nevirapin 1 μΜ, Boehringer Ingelheim) and this baseline subtracted from fluorescence signal. Non-specific fluorescence due to secondary transfection caused by co-precipitation of DNA by calcium / phosphate in the vector supernatants was eliminated by treatment of the vector stock with DNasel prior to transduction.

Under these conditions, the presence of the triplex sequence in the HIV vector increased the GFP transduction in HeLa cells by more than ten fold (Figure 5B). A similar increase of gene transduction was observed in other cell lines such as MT4 or 293T (data not shown). This effect was lost if the triplex sequence was inserted in reverse orientation (Figure 5B) or mutated in cPPT (not shown).

Example 9: The presence of a triplex DNA in HIV-1 vectors increases the vector genome nuclear import rate to wild-type levels

It was then of interest to determine whether the increase in GFP fluorescence induced by insertion of a triplex sequence into the HIV vector was due to its effect on nuclear import of DNA vector. To address this issue, we adapted our quantitative Southern blot assay to intracellular viral DNA in the vector system. DNA from transduced cells per vector was digested with EcoNI and Avall to produce an internal 0.8 kb fragment, and with XhoI. Using a PCR-generated DNA probe overlapping accurately the EcoNI site, specific signals were expected for the non-integrated linear vector genome, and for one and two 1.2 kb, 1.4 kb circular DNA LTRs, and 2 kb respectively. The DNA vector processing was analyzed at various times after HeLa cell transduction. The total amount of DNA vector synthesized in transduced cells was comparable for vectors containing or lacking the triplex DNA (Figure 5C). Again, insertion of the cPPT and CTS sequences in any of the orientations into the HR vector did not influence the reverse transcription rate of their genome. After quantification by &quot; phosphorimager &quot;, we found the intracellular DNA target of the HR-GFP and TRIPinv-GFP vectors (Figure 5D) to closely resemble the central triplex DNA of defective viruses and the TRIP-GFP DNA vector in resembling DNA from the wild type HIV-1 LAI virus (Figure 3C).

A defect in nuclear DNA import was evident in the case of the HR-GFP and TRIPinv-GFP vectors. The intracellular DNA of these vectors was characterized by a strong accumulation of nonintegrated linear molecules, together with small amounts of integrated proviur and one and two circular LTRs. This DNA profile strongly resembled that of the cPPT-D mutant virus. At completion of DNA vector processing in transduced cells, 70 to 80% of the HR-GFP and TRIPinv-GFP element DNA remained in the form of non-integrated linear molecules, while only 10 to 15% were present as non-integrated in circular LTR and 5 to 10% as integrated proviruses. This low but detectable amount of integrated DNA vector would respond by gene transduction obtained using HR-GFP or TRIPinv-GFP vectors. 48

This quantitative assay also showed that insertion of the HIV triplex DNA sequence into the HR vector in the correct orientation complemented its nuclear import deficiency at wild-type levels. The final state of the TRIP-GFP DNA in transduced cells was similar to that observed with wild-type HIV-1 virus: 50% or more of the DNA vector was integrated into the target cell chromatin, a major fraction was circularized and some molecules remained as DNA (Figure 5D and 3C). Conversely, insertion of the triplex sequence into the HR vector upstream of the internal CMV promoter did not influence GFP expression at the transcriptional level. This was conferred by transfection of HeLa cells with plasmids pHR-GFP, pTRIP-GFP, and pTRIPinv-GFP and quantification by fluorescence (data not shown).

It can be deduced from these results that the increase in GFP transduction obtained with the TRIP-GFP vector is completely attributable to the strong stimulation of its nuclear import by the presence of the triplex. This finding again highlights the important role of HIV triplex DNA in the nuclear import of viral and vector DNA.

Example 10: HIV-1 vectors containing the triplex DNA sequence allow efficient gene transfer in hematopoietic stem cells

Figures 7A and 7B illustrate the results of two successful transduction experiments using human cord CD34 + cells. They show that after a short transduction protocol of 24 or 60 hours, respectively 71.5% and more than 90% of CD34 + cells strongly express the GFP reporter protein. This expression reflects stable cell transduction since viral integration was confirmed, at least in the same proportions, by PCR assays in DNA extracted from parent colon-derived cell colonies 14 days after sowing cells in clonogenic progenitor assay. Identical results were obtained using 49 freshly purified CD34 + cells or the same CD34 + cells that were frozen after purification and thawed several weeks later for the transduction experiment. We also obtained comparable transduction efficiencies using another source of hematopoietic stem cells, mobilized peripheral blood stem cells (PBMC), collected by &quot; cytapheresis &quot; after stimulation by cytokine. CD34 + PBMCs were also transduced immediately after purification or after a freeze / thaw step with identical results. Using long-term culture (LTC) and NOD-SCID repopulation assays we have shown that we transduced cells having multiple lympho-myeloid potentials and the ability to repopulate NOD-SCID mouse bone marrow 4 months after grafting. These functional assays represent the latest experiments currently available to evaluate the function of human hematopoietic cells.

Example 11: The presence of the triplex DNA sequence in lentiviral vector elements strongly stimulates gene transfer to hematopoietic stem cells The dose / response experiment shown in Figure 8 (representative of 3 experiments) was designed to compare transduction efficiency in human hematopoietic stem cells of HIV-1 vectors including or lacking the triplex DNA sequence. We first plotted (A) the percentage of CD34 + eGFP + cells obtained as a function of the vector concentration used for transduction. We observed that, regardless of the vector dose, the TRIP + vector was more efficient than TRIP, with a mean of 40 ± 19% and 15.4 ± 12.5% of the CD34 + cells respectively being eGFP + for 500 ng P24 viral / ml of each vector = 3 exp). A 4-6 fold increase in the final percentage of GFP + cells was obtained after TRIP-GFP vector transduction when compared to results obtained after HR-GFP vector transduction. The difference in efficiency between the two vectors is also enhanced when the mean fluorescence intensity is plotted against the virus dose. It is reached on the plateau for the TRIP- (HR-GFP) vector at doses of 100ng P24 / ml whereas the intensity of fluorescence in transduced cells increases with the dose of TRIP + vector. This could reflect the limitation on nuclear import of the pre-integrative forms of the TRIP vector and the increasing number of cell-integrated copies after transduction with increasing doses of the TRIP-GFP vector. The third graphic integrates both aspects and shows the resulting effect of the triplex DNA sequence on GFP fluorescence activity in human HSC. As shown, the presence of the triplex DNA sequence in the HIV vector induces increased production of GFP in HSC by a factor of more than ten-fold.

Example 12: An integrated copy fraction of HIV vector genomes remains silent on human hematopoietic transduced cells Inactivation of the integrated transgene may occur. Transfer efficiency was always better when evaluated by percentage of transduced progenitor-derived cell colonies determined by PCR assay for the integrated lentiviral vector rather than when evaluated by the percentage of CD34 + / eGFP + cells determined by FACS analysis 48 hours after the end of the transduction protocol. This reflects the occurrence of transcriptionally active proviruses either due to their site of integration or to the progressive and random inactivation of the provirus while the cells proliferate and differentiate. We observed in colonies derived from a single clonal myelogenous progenitor that some of the subclones could be GFP luminous while others were negative, reflecting the random transcriptional inactivation of the transgene integrated in this phenotypically homogenous clonal progeny.

Example 13: HIV vectors containing a deleted version of the U3 region of LTR and an internal EFla are more potent systems for the transduction of hematopoietic stem cells 51

In Figure 9, we compared the ability of various HIV-1 derived vectors, including the triplex DNA sequence, to transduce CD34 + cells from human cord blood. The deletion effect of most of the U3 region of 3'LTR on GFP transduction and expression in CD34 + cells was analyzed. Comparison of vectors containing an intact HIV-1 LTR or a deleted version of U3 was conducted in the context of a CMV internal promoter or an internal EFIa promoter (Kim et al., 1990) to conduct expression of the GFP reporter gene . All transduction experiments were conducted using the same concentration of vector particles (500 ng P24 / ml) after normalization of vector stocks using a commercially available ELISA assay for P24 HIV-1 antigen (Dupont) (capsid protein ). Flow cytometry analysis (FACS) was performed either 48 hours (Figure 9A) or 120 hours (Figure 9B) after the 24 hour transduction period.

Interestingly, the deletion of the U3 region of the LTR in HIV-1 vectors induced a slight increase in the percentage of GFP positive cells in all cases. This increase was modest when analyzed after 48 hours. At this point, a pseudotransduction mechanism may be responsible for a fraction of the GFP-positive cells. Pseudotransduction is the direct delivery of GFP proteins to target cells by the retroviral vector particle fusing, without the need for a current integration of the retroviral vector genome. The increase in the percentage of positive GFP after transduction by the deleted versions of U3 from the HIV-1 vectors became more evident 120 hours after transduction (Figure 9B). At this point, up to two-fold increase in the percentage of GFP positive cells following transduction by the TRIP AU3-EFI-GFP vector is observed when compared to the results obtained with the equivalent vector but containing an intact HIV-1 LTR. 52

More importantly, the deletion of the U3 region of the HIV-1 LTR in the HIV-1 triplex vectors induced better expression of the GFP reporter protein. The mean fluorescence intensity in human HSC transduced when analyzed by FACS was always greater by a factor of three to five times in the case of the deleted versions of U3 than with vectors containing an intact HIV-1 LTR. This benefit of GFP expression was observed whether the CMV or EFla promoters were used as the internal promoter in the HIV-1 vector element. The molecular mechanism explaining this increased expression of GFP proteins in transduced cells is not known. Some sequence in HIV-1 LTR can negatively influence expression driven by the internal promoter. Alternatively, a baseline transcription initiated on the HIV-1 LTR may interfere with transcriptional initiation in the internal promoter.

This study also shows that the EFla promoter is a better promoter in human HSC than the CMV promoter. In Figure 9B, the mean GFP fluorescence intensity is three to five times better in the case of the EFla promoter than in the case of the CMV promoter. 53

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LIST OF SEQUENCES

&lt; 110 &gt; INSTITUTE PASTEUR INSERM &lt; 120 &gt; LENTIVIRAL TRIPLEX DNA, AND VECTORS AND RECOMBINANT CELLS CONTAINING LENTIVIRAL TRIPLEX DNA &lt; 130 &gt; B4681_01.app &lt; 140 &gt; STILL UNCONSCIOUS &lt; 141 &gt; 2000-10-10 &lt; 150 &gt; 60 / 158,387 &lt; 151 &gt; 1999-10-12 &lt; 160 &gt; 24 &lt; 170 &gt; Patentln Ver. 2.1

&lt; 210 &gt; 1 &lt; 211 &gt; 25 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt; &lt; 223 &gt; Description of Artificial Sequence: PLASMIDAL BASED MUTAGENESIS INITIATOR pLAI3 &lt; 400 &gt; 1 caattttaaa agaagagggg ggatt 25

&lt; 210 &gt; 2 &lt; 211 &gt; 43 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt; &lt; 223 &gt; Description of Artificial Sequence: PLASMIDAL BASED MUTAGENESIS INITIATOR pLAI3 &lt; 400 &gt; 2 attcatccac aacttcaagc gccgcggtgg tattgggggg tac 43

&lt; 210 &gt; 3 &lt; 211 &gt; 23 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt;

&lt; 223 &gt; Description of Artificial Sequence: INITIATOR TO AMPLIFY NUCLEIC ACID CODING GREEN FLUORESCENT PROTEIN INCREASED &lt; 400 &gt; 3 ccggatcccc accggtcgcc acc 23

&lt; 210 &gt; 4 &lt; 211 &gt; 23 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt;

&lt; 223 &gt; Description of Artificial Sequence: NUCLEOTIDE AMPLIFIER INITIATOR THAT ENHANCES GREEN FLUORESCENT PROTEIN INCREASED &lt; 400 &gt; 4 ccctcgagct agagtsgcgg ccg ??? 23

&lt; 210 &gt; 5 &lt; 211 &gt; 47 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt;

&lt; 223 &gt; Description of Artificial Sequence: INITIATOR TO AMPLIFY PUCLTRRI &lt; 400 &gt; 5 cggaattcgg atccgcggcc gcatcgatct tgtcttcgtt gggagtg 47

&lt; 210 &gt; 6 &lt; 211 &gt; 40 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt;

&lt; 223 &gt; Description of Artificial Sequence: INITIATOR TO AMPLIFY PUCLTRRI &lt; 400 &gt; 6 cggaattcag ccgtctcgag agatgctgca tataagcagc 40

&lt; 210 &gt; 7 &lt; 211 &gt; 38 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt; &lt; 223 &gt; Description of Artificial Sequence: INITIATOR TO AMPLIFY cPPT AND CTS OF pLAI3 &lt; 400 &gt; 7 gtggtcggcg ccgaattcac aaatggcagt attcatcc 38

&lt; 210 &gt; 8 &lt; 211 &gt; 34 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt; 65 &lt; 223 &gt; Description of Artificial Sequence: INITIATOR TO AMPLIFY cPPT AND CTS OF pLAI3 &lt; 400 &gt; 8 gtcgtcggcg ccccaaagtg gatctctgct gtcc 34

&lt; 210 &gt; 9 &lt; 211 &gt; 38 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt;

&lt; 223 &gt; Description of Artificial Sequence: INITIATOR TO AMPLIFY TRIPLEX SEQUENCE OF THE EF1 alpha ROMOTOR IN MATRIX pLai &lt; 400 &gt; 9 gtcgtcggcg ccgaattcac aaatggcagt attcatcc 38

&lt; 210 &gt; 10 &lt; 211 &gt; 39 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt;

&lt; 223 &gt; Description of Artificial Sequence: INITIATOR TO AMPLIFY TRIPLEX SEQUENCE OF EF1 alpha PROMOTER IN MATRIX pLai &lt; 400 &gt; 10 agcctcacga cgcgtatcag ccaaagtgga tctctgctg 39

&lt; 210 &gt; 11 &lt; 211 &gt; 26 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt;

&lt; 223 &gt; Description of Artificial Sequence: INITIATOR TO AMPLIFY TRIPLEX SEQUENCE OF EF1 alpha PROMOTER IN MATRIX pEFpgkneo &lt; 400 &gt; 11 ctgatacgcg tcgtgaggct ccggtg ??? 26

&lt; 210 &gt; 12 &lt; 211 &gt; 26 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt;

&lt; 223 &gt; Description of Artificial Sequence: INITIATOR TO AMPLIFY TRIPLEX SEQUENCE OF EF1 alpha PROMOTER IN MATRIX pEFpgkneo &lt; 400 &gt; 12 cgggatcctg tgttctggcg gcaaac 26 &lt; 210 &gt; 13 &lt; 211 &gt; 23

&lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 13 2 23 ccctcgagct agagtcgcgg ccg &lt; 210 &gt; 14 &lt; 211 &gt; 23

&lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 14 ccggatcccc accggtcgcc acc 67

&lt; 210 &gt; 15 &lt; 211 &gt; 21 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt;

&lt; 223 &gt; Description of Artificial Sequence: INITIATOR FOR AMPLIFICATION OF pLAI3R VIRAL DNA &lt; 400 &gt; 15 agaagaaatg atgacagcat g ??? 21

&lt; 210 &gt; 16 &lt; 211 &gt; 17 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt;

&lt; 223 &gt; Description of Artificial Sequence: INITIATOR FOR AMPLIFICATION OF pLAI3R VIRAL DNA &lt; 400 &gt; 16 tgccagttct agctctg 17

&lt; 210 &gt; 17 &lt; 211 &gt; 20 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt;

&lt; 223 &gt; Description of Artificial Sequence: INITIATOR FOR PROBE SYNTHESIS FOR VECTOR pTRIPGFP &lt; 400 &gt; 17 cagggacttg aaagcgaaag 20 &lt; 210 &gt; 18

&lt; 211 &gt; 27 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt;

&lt; 223 &gt; Description of Artificial Sequence: INITIATOR FOR PROBE SYNTHESIS FOR VECTOR pTRIPGFP &lt; 400 &gt; 18 gcttgtgtaa ttgttaattt ctctgtc 27 &lt; 210 &gt; 19 &lt; 211 &gt; 7

&lt; 212 &gt; PRT &lt; 213 &gt; Human immunodeficiency virus type 1 &lt; 220 &gt; &lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1) .. (7) &lt; 223 &gt; Partial HIV-1 cPPT sequence &lt; 400 &gt; 19

Asn Fen Lys Arg Lys Gly Gly I 5

&lt; 210 &gt; 20 &lt; 211 &gt; 19 &lt; 212 &gt; DNA &lt; 213 &gt; Human immunodeficiency virus type 1 &lt; 400 &gt; 20 ttttaaaaga aaagggggg

&lt; 210 &gt; 21 &lt; 211 &gt; 19 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence 69 &lt; 220 &gt; &lt; 223 &gt; Description of Artificial Sequence: MUTATION IN SEQUENCE cPPT OF HIV-1 &lt; 400 &gt; 21 ttttaaacgc aaaggtggt

&lt; 210 &gt; 22 &lt; 211 &gt; 7 &lt; 212 &gt; PRT &lt; 213 &gt; Artificial Sequence &lt; 220 &gt; &lt; 223 &gt; Artificial Sequence Sequence: HIV-1 cPPT SEQUENCE PEPTIDE &lt; 400 &gt; 22

Asn Fen Lys Arg Arg Gly Gly I 5

&lt; 210 &gt; 23 &lt; 211 &gt; 19 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt;

&lt; 223 &gt; Description of Artificial Sequence: MUTATION IN SEQUENCE OF HIV cPPT CODIFICATION &lt; 400 &gt; 23 ttttaaaaga agagggggg

&lt; 210 &gt; 24 &lt; 211 &gt; 19 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence

INTRODUCED 19

MUTANT NA INTRODUCED 1 19 70 &lt; 220 &gt; &lt; 223 &gt; Description of Artificial Sequence: INTRODUCED HIV-1 cPPT CODE CHANGE &lt; 400 &gt; 24 cttcaagcgc cgcggtggt 19 17-04-2007 71

Claims (32)

An HIV-1 expression vector comprising: a) an isolated or purified nucleic acid comprising at least one copy of the cPPT and CTS cis-actuation regions of a retrovirus, wherein said cPPT and CTS regions induce a DNA structure and a heterologous nucleic acid sequence, wherein the HIV-1 LTR is deleted for the promoter and the U3 enhancer. The vector of claim 1 wherein said nucleic acid comprising at least one copy of the cPPT and CTS regions is a lentivirus. The vector of claim 2 wherein said nucleic acid comprising at least one copy of the cPPT and CTS regions is human immunodeficiency virus (HIV). The vector of claim 3, wherein said nucleic acid comprising at least one copy of the cPPT and CTS regions is either HIV-1 or HIV-2. The vector of claim 2, wherein said nucleic acid comprising at least one copy of the cPPT and CTS regions is from VISNA, EIAV, FIV, or CAEV. The vector of claims 1 to 5, comprising a single copy of the cPPT and CTS regions of the retrovirus. A vector according to any one of claims 1 to 6, wherein the heterologous nucleic acid sequence is operably or physically linked to the nucleic acid comprising at least one copy of the cPPT and CTS regions. 1 A vector according to any one of claims 1 to 7, wherein the heterologous nucleic acid sequence encodes a peptide, polypeptide, or protein. The vector of claim 8 containing a copy of the cPPT-CTS cis-acting regions, the GFP reporter gene under the control of the EFla promoter, and the U3 sequence of an LTR, deprived of its promoter and enhancer, and which is pTR1P Δυ3 EFlaGFP. The vector of claim 8, wherein the heterologous nucleic acid sequence encodes a therapeutic protein. A recombinant cell comprising an expression vector according to any one of claims 1 to 10. The recombinant cell of claim 11, wherein the cell is a HeLa cell or a hematopoietic cell. The recombinant cell of claim 11, which is a hematopoietic stem cell. An in vitro process for inserting a heterologous nucleic acid sequence into the nucleus of a target cell, said method comprising exposing an expression vector according to any one of claims 1 to 10 under conditions allowing the uptake of said acid sequence nucleic acids in the target cell. The process of claim 14, wherein the insertion efficiency of the nucleic acid of interest in the target cell nucleus is 30% or greater. A method according to any one of claims 14 to 15, wherein the heterologous nucleic acid encodes a peptide, polypeptide, or protein. 2 The process of claim 16, wherein the protein is a therapeutic protein. A method according to any one of claims 14 to 17, wherein the target cell is a non-dividing cell. A method according to any one of claims 14 to 17, wherein the target cell is a HeLa cell or a hematopoietic cell. A method according to any one of claims 14 to 17, wherein the target cell is a hematopoietic stem cell. A method for expressing a heterologous nucleic acid sequence in vitro, said method comprising: a) exposing target cells to an expression vector according to any one of claims 1 to 10, under conditions that allow for the uptake of the vector containing the sequence of heterologous nucleic acids in the target cell to create a recombinant cell, and b) culturing the recombinant cell under conditions where the heterologous nucleic acid sequence is expressed. The process of claim 21, wherein the heterologous nucleic acid sequence is expressed in tissue culture. A method according to any one of claims 21 to 22, which further comprises purifying or isolating the expression product of the heterologous nucleic acid sequence. Recombinant cell comprising an expression vector according to any one of claims 1 to 10 for use as a therapeutic compound to allow the recombinant cell to express the nucleic acid in the subject's body. 3 The recombinant cell of claim 24 for use as a therapeutic compound in a hematopoietic stem cell. An expression vector according to any one of claims 1 to 10 for use as a therapeutic compound, wherein the nucleic acid is in an amount and form sufficient to result in the expression of the gene of interest in the subject's body. The vector of claim 26, which is expressed in a target tissue. An expression HIV-1 vector comprising: a) an isolated or purified nucleic acid comprising at least one copy of the cPPT and CTS cis-actuation regions of a retrovirus, wherein said cPPT and CTS regions induce a DNA structure of and a heterologous nucleic acid sequence, wherein the HIV-1 LTR is deleted for the promoter and the U3 enhancer, for use in the treatment of an individual suffering from, or having a high probability of developing, a disease or disorder having a genetic basis, wherein said retroviral vector is in an amount sufficient to result in the expression of said therapeutic protein in an amount sufficient to treat said disease or disorder. The vector of claim 28 for use as a drug in a prophylactic, improvement or curative treatment. The vector of claim 29, for use in the treatment of a disease or disorder of the blood, a disease or disorder of the brain or nervous system, or a developing disease or disorder. 4 A kit containing at least one container containing an expression vector according to any one of claims 1 to 10. The kit of claim 31, wherein the heterologous nucleic acid sequence encodes a therapeutic protein. 4/17/2007